IGEM:MIT/2007/Notebook/2007-7-5

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Contents

Agenda

  1. Prepare chemically competent cells from the DH5a line
  2. Make long term glycerol stock of DH5a

DH5a Incubation

  • both 10 mL overnight cultures had growth

Making long term glycerol stock of DH5a

  • Followed OWW protocol: http://www.openwetware.org/wiki/Endy:Making_a_long_term_stock_of_bacteria
  • Method:
    • Add 1 ml of 40% glycerol in H2O to a cryogenic vial.
    • Add 1 ml sample from the culture of bacteria to be stored.
    • Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed.
      • Alternatively, pipet to mix.
    • Use a tough spot to put the name of the strain or some useful identifier on the top of the vial.
    • On the side of the vial list all relevant information - part, vector, strain, date, researcher, etc.
    • Store in a freezer box in a -80C freezer. Remember to record where the vial is stored for fast retrieval later.

Preparing Chemically Competent Cells

  1. Grow an overnight culture of cells in LB media. In the morning, dilute 2mL of culture back into 198ml of fresh LB media in a 200ml conical flask (Repeat for remaining amounts). You should aim to dilute the overnight culture by at least 1/100.
  2. Grow the diluted culture to an OD600 of 0.2 - 0.5. (You will get a very small pellet if you grow 25ml to OD600 0.2)
  3. Put eppendorf tubes on ice now so that they are cold when cells are aliquoted into them later. If your culture is X ml, you will need X tubes. At this point you should also make sure that your TSS is being chilled (it should be stored at 4oC but if you have just made it fresh then put it in an ice bath).
  4. Split the culture into two 50ml falcon tubes and incubate on ice for 10 min.

All subsequent steps should be carried out at 4oC and the cells should be kept on ice wherever possible

  1. Centrifuge for 10 minutes at 3000 rpm and 4oC.
  2. Remove supernatant. The cell pellets should be sufficiently solid that you can just pour off the supernatant if you are careful. Pipette out any remaining media.
  3. Resuspend in chilled TSS buffer. The volume of TSS to use is 10% of the culture volume that you spun down. You may need to vortex gently to fully resuspend the culture, keep an eye out for small cell aggregates even after the pellet is completely off the wall.
  4. Add 100 μl aliquots to your chilled eppendorfs and store at − 80oC.
  • The original paper [1] suggests freezing the cells immediately using a dry ice bath. I (BC) have used liquid nitrogen quite successfully instead of dry ice. Simply placing the cells at − 80oC also seems to work well (Jkm)
  • It is a good idea to run a positive control on the cells.
    • The Endy Lab is trying to use a standard positive control to better compare (and hopefully improve) the transformation efficiencies in the lab, you can check it out here.

Transform DH5a with F2620, RBS B0034, and eRFP E1010

  1. Find location of dry DNA in BioBrick Wells
  2. Add 15µl H20 to dry DNA in well
  3. Pipette out into PCR tube and freeze in -20C to store
  4. Add 1µl of hydrated DNA for each transformation in eppendorf tube
  5. Add 150µl of competent cells
  6. Pipette gently to mix
  7. Let mix sit for 30 min. on ice
  8. Heat shock - incubate for 20 seconds at 37C
  9. Incubate cells on ice for 2 min.
  10. Add 1mL LB at room temp.
  11. Incubate for 1 hr. at 37C on shaker
  12. Spread 150µl onto plate with appropriate antibiotic
  13. Grow overnight at 37C
  14. Save rest of transformant in liquid culture at 4C.

Plates

  • eRFP: (#1) 200 µl; (#2) 200 µl; (#3) 150 µl
  • F2620: (#1) 150 µl; (#2) 150 µl; (#3) 150 µl
  • B0034: (#1) 150 µl; (#2) 150 µl; (#3) 120 µl
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